1. Field of Invention
The present invention relates generally to cellular therapy and cell encapsulation, and more specifically, to techniques for loading a patient implantable encapsulation device with biological material such as, but not limited to insulin producing pancreatic cells developed from allogeneic pluripotent human stem cells, such as embryonic stem (hES) cells for the treatment of diabetes mellitus.
2. Description of Related Art
Nearly 25 million people in the United States are afflicted with diabetes mellitus, which is a disease caused by the loss of the ability to transport glucose into the cells of the body, because of either a lack of insulin production (commonly known as “Type 1” diabetes, insulin-dependent diabetes, or juvenile diabetes) or diminished insulin response (commonly known as “Type 2” diabetes). Type 1 diabetes is characterized by high blood sugar from loss of insulin producing pancreatic beta cells, leading to insulin deficiency and poor blood sugar regulation. Type 1 diabetes can result in serious complications if left untreated, such as cardiovascular disease, retinal damage, and even death. Type 1 diabetes usually cannot be cured, and has historically been managed with subcutaneous insulin injections from a syringe or an insulin pump. However, multiple daily injections of insulin do not adequately mimic the body's minute-to-minute production of insulin and precise control of glucose metabolism. Mortality and morbidity still occur today with insulin treatment from over dosage of insulin, which results in extreme hypoglycemia (low blood sugar) and coma followed by death unless reversed by someone who can quickly administer glucose to the patient. Extreme under dosage of insulin, leading to hyperglycemia (high blood sugar) and ketoacidosis can also result in coma and death if not properly and urgently treated. Even with insulin therapy, the average life expectancy of a diabetic is 15-20 years less than a healthy person.
Type 2 diabetes usually appears in middle age or later, and particularly affects those who are overweight. Over the past few years, however, the incidence of Type 2 diabetes in young adults has increased dramatically. In Type 2 diabetes, cells that normally respond to insulin lose their hormone sensitivity and fail to respond to insulin normally. This insulin resistance may be overcome for many years by extra insulin production by the pancreatic beta cells. Over time, the beta cells may become exhausted due to the burden of producing large amounts of excess insulin in response to elevated blood glucose levels. Ultimately, the overworked beta cells die and insulin secretion fails, bringing with it a concomitant rise in blood glucose to sufficient levels that it can only be controlled by exogeneous insulin injections. High blood pressure and abnormal cholesterol levels usually accompany Type 2 diabetes. These conditions, together with high blood sugar, increase the risk of heart attack, stroke, and circulatory deterioration, which in the legs can necessitate amputation. Drugs to treat Type 2 diabetes include some that act to reduce glucose absorption from the gut or glucose production by the liver, others that reduce the formation of more glucose by the liver and muscle cells, and still others that stimulate the beta cells directly to produce more insulin. Nevertheless, high levels of glucose are toxic to beta cells, causing a progressive decline of function and cell death, despite pharmacological interventions. Consequently, many patients with Type 2 diabetes eventually need exogenous insulin.
Another form of diabetes is called Maturity Onset Diabetes of the Young (MODY). This form of diabetes is due to one of several genetic errors in insulin-producing cells that restrict their ability to process the glucose that enters via special glucose receptors. Beta cells in patients with MODY cannot produce insulin correctly in response to glucose, which results in hyperglycemia. The patient's treatment eventually leads to the requirement for insulin injections.
The currently available medical treatments for patients that require exogenous insulin are limited to insulin administration and transplantation of either whole pancreata or pancreatic segments.
Insulin therapy is far more prevalent than pancreas transplantation. Insulin administration is managed conventionally by a few daily or weekly blood glucose measurements and subcutaneous injections; intensively by multiple blood glucose measurements and multiple subcutaneous injections of insulin; or continuously by subcutaneous injections of insulin with a pump. Conventional insulin therapy involves the administration of one or two injections per day of intermediate-acting insulin with or without the addition of small amounts of regular insulin. Intensive insulin therapy involves multiple administrations of intermediate- or long-acting insulin throughout the day, combined with regular or short-acting insulin prior to each meal. Continuous insulin administration involves the use of a small battery-driven pump that delivers insulin subcutaneously to the abdominal wall, usually through a 27-gauge butterfly needle. This treatment modality provides continuous insulin delivery at a basal rate throughout the day and night, with increased amounts or boluses programmed prior to meals. In each of these methods, the patient is required to frequently monitor his or her blood glucose levels and, if necessary, adjust the insulin dose. However, controlling blood sugar is not simple. Despite rigorous attention to maintaining a healthy diet, exercise regimen, and scrupulous attention to proper dosing of insulin, many other factors can adversely affect a person's blood-sugar including stress, hormonal changes, periods of growth, illness, infection and fatigue. Insulin-dependent diabetes is a chronic, life threatening disease, which requires constant vigilance.
Ultimately, it is the goal of research efforts to regenerate pancreatic insulin-producing cells in the body or repopulate these cells in situ. Although that is not possible at this time, it may eventually be feasible to transplant cells that produce insulin or precursors that will differentiate into insulin producing cells. It is likely that such cells will eventually die rather than recreate the self perpetuating system of pancreatic insulin producing cell development. Thus, they will need to be replaced periodically. In addition, exogenous cell transplantation carries the risk of introducing undesirable cell populations that may be pathogenic. Of particular concern is the possibility that a patient will receive cells capable of forming tumor in the human body. Thus, transplantation of insulin-producing cells or precursors is best performed using encapsulation forms of cells. Encapsulation permits subsequent removal of cells that are no longer therapeutically effective while reducing the risk of unwanted cell growth in the body. Furthermore, encapsulation can protect the transplant from attack by the patient's own immune system, which can destroy the transplanted cells in a short time if unabated.
Encapsulation of cells for the potential of treating a number of diseases and disorders has been discussed in the literature. The concept was suggested as early as 100 years ago, but little scientific research was performed prior to the 1950's when immunologists began using cells encapsulated within membrane devices to separate implanted cells from host cells to better understand certain aspects of the immune system. Cell encapsulation technology has potential applications in many areas of medicine. For example, in addition to treatment of diabetes (Goosen et al. (1985) Biotechnology and Bioengineering, 27:146), applications include production of biologically important chemicals (Omata et al. (1979) “Transformation of Steroids by Gel-Entrapped Nacardia rhodocrous Cells in Organic Solvent” Eur. J. Appl. Microbiol. Biotechnol. 8:143-155), and evaluation of anti-human immunodeficiency virus drugs (McMahon et al. (1990) J. Nat. Cancer Inst., 82(22) 1761-1765).
There are three main types of encapsulated devices categorized by form of encapsulation: 1) macrodevices, 2) microcapsules, and 3) conformal coatings.
Macrodevices are larger devices containing membranes in the form of sheets or tubes, and usually include supporting structures. Two major types of macrodevices have been developed: a) flat sheet and b) hollow fiber.
Among the flat sheet devices, one type (Baxter, Theracyte) is made of several layers for strength and has diffusion membranes between support structures with loading ports for replacing the cells. This device form is generally most suitable for encapsulation of insulin-producing cells.
The other important macrodevice type is the hollow fiber, made by extruding thermoplastic materials into hollow fibers. These hollow fibers can be made large enough to act as blood conduits. However, due to low packing densities, the required cell mass for clinical human dose causes the length of this type of hollow to approach many meters. Therefore, this approach has largely been abandoned for treating diabetes.
The microcapsule was one of the first to devices promising potential clinical efficacy. A microcapsule's function is to protect the graft with a membrane permeable to glucose and insulin, but impermeable to components of the immune system. One of the problems associated with microcapsules is their relatively large size in combination with low packing densities of cells, especially for the treatment of diabetes. In addition, many of the molecules used to produce microcapsules may cause an inflammatory reaction or may also be reactive within the host after implantation.
The last category of cell encapsulation is conformal coating. A conformally coated cell aggregate is one that has a substantially uniform cell coating around a cell aggregate regardless of size or shape of the aggregate. This coating not only may be uniform in thickness, but it also may be uniform in the protective permselective nature of the coating that provides uniform immunoisolation. Furthermore, it may be uniform in strength and stability, thus preventing the coated material from being violated by the host's immune system.
An important aspect to the feasibility of using these various methods of encapsulating cells for implantation is the relevant size and implant site needed to obtain a physiological result. For diabetes treatment, production of 5,000 IEQ/kg-BW of insulin is required. Injecting isolated islets into the hepatic portal vein requires 2-3 ml of packed cells to achieve this therapeutic level of insulin production. A macro-device consisting of a flat sheet that is 1 islet thick (˜200 μm) requires a surface area equivalent to 2 US dollar bills. A macro-device consisting of hollow fibers with a loading density of 5% would need 30 meters of fiber. Alginate microcapsules with an average diameter of 400-600 μm would need a volume of 50-170 ml.
The stringent requirements for encapsulation polymers of biocompatibility, chemical stability, immunoprotection and resistance to cellular overgrowth restrict the applicability of existing methods of encapsulating cells and other biological materials. Due to the inability of those of skill in the art to provide all the essential properties of successful cell encapsulation, none of the encapsulation technologies developed in the past have resulted in a clinical product. These properties can be broken down into the following categories:
Biocompatibility—The materials used to make an encapsulating device must not elicit a host response, which may cause a non-specific activation of the immune system by these materials alone. When considering immunoisolation, one must recognize that it is optimal if there is minimal activation of the host immune cells in response to the materials. If there is activation of the host immune cells by the materials, then the responding immune cells will surround the device and attempt to destroy it. This process may produce cytokines that will certainly diffuse through the capsule and may destroy the encapsulated cells. Most devices tested to date have failed in part from their lack of biocompatibility in the host.
Porosity—There exists an important balance between having the largest pores possible in the barrier surrounding encapsulated cells to permit nutrients, waste materials and therapeutic products to pass through, and having the smallest pore size required to both retain cells and keep elements of the immune system segregated from the encapsulated cells. The optimal cell encapsulation barrier has an exact and consistent porosity, which allows maximal cell survival and function, as well as isolation from the host immune response.
Encapsulated Cell Viability and Function—Encapsulating materials should not be cytotoxic to the encapsulated cells either during the formation of the coatings or thereafter, otherwise the number of encapsulated cells will decrease and risk falling short of a therapeutically effective dose.
Relevant Size—Many devices are of such a large size that the number of practical implantation sites in the host is limited. Furthermore, relative diffusion distance between the encapsulated cells and the host is increased with increasing device size. The most critical diffusive agent for cell survival is oxygen, which requires minimal diffusion distances because the starting partial pressure of oxygen is already low at the tissue level in the body.
Cell Retrieval or Replacement—The encapsulating device should be retrievable, refillable, or biodegradable, allowing for replacement or replenishment of the cells. Many device designs have not considered the fact that encapsulated cells have a limited lifetime in the host and require regular replacement.
Therapeutic Effect—The implant should contain sufficient numbers of functional cells to have a therapeutic effect for the disease application in the host.
Clinical Relevance—The encapsulating cell device should have a total volume or size that allows it to be implanted in the least invasive or most physiologically relevant site for function, and which has a risk/benefit ratio below that faced by the host with the current disease or disorder.
Commercial Relevance—The encapsulating cell device should be able to meet the above requirements in order for it to be produced on an ongoing basis for the long-term treatment of the disease process for which it has been designed.
All of the above factors must be taken into consideration when evaluating a specific technique, method or product for use in implantation of insulin-producing cells to alleviate the effects of diabetes.
Transplantation of human islets with immunosuppression can be performed by introducing unencapsulated islets directly via percutaneous injection between the ribs, through the liver, and into the portal vein using fluoroscopic guidance with an introduction catheter. Essentially all of the human islet transplants have been performed using this technique. A major risk of this procedure is increased portal venous pressures depending on the rate of infusion and the amount infused. Additional risk is associated with injection of islet tissues insufficiently purified, which can also lead to portal venous thrombosis. As the interventional radiologist prepares to withdraw the catheter, a bolus of gelatin is left behind to prevent hemorrhaging from the injection site. Unfortunately, several patients have had bleeding episodes following this procedure.
In addition to injecting the islets into the portal vein, a few patients have had islets injected into the body of the spleen. The spleen is more fragile than the liver so these injections have typically been performed at the time of, e.g., kidney transplantation, thus permitting splenic injection as an open procedure. Freely injecting islets into the peritoneal cavity has been performed in mice without difficulty. In using this site in larger animals or humans, it has been found that twice the number of islets is needed if injected into the peritoneal cavity than required in the portal vein implants. If any rejection or inflammatory reactions occur, then adhesions tend to form between the loops of intestine, as well as, to the omentum. This reaction can lead to additional problems long term, such as bowel obstruction. Thus, the ability to implant encapsulated islets or other insulin-producing cells into a subcutaneous site would significantly reduce the complications associated with these other procedures and modalities.
Before any type of encapsulation device is implanted in a patient, it must be carefully loaded with cells, which has been conventionally performed manually by a skilled technician. Typically, encapsulation devices are loaded directly using positive pressure from a syringe. The technician fills the syringe with cells, and then inserts the syringe's needle into an inlet port of the encapsulation device, while the encapsulation device is outside the patient. The syringe exerts positive pressure on the cells to force the cells into the encapsulation device. The encapsulation device is then sealed and is later implanted into the patient.
However, loading an encapsulation device directly from a syringe has several drawbacks in both safety and cell viability. Cells often leak from the device when the syringe is removed from the port. The syringe's needle can also pierce the wall of the encapsulation device, permitting contamination of the outside of the encapsulating device with cells during loading or after implantation in the patient. Such contamination is a safety hazard regulated by the U.S. Food and Drug Administration. Theoretically, even a single contaminating cell could expand and/or biodistribute. The syringe also creates high positive pressure in the needle, which can cause shear stress and decrease cell viability.